"
Team:TUDelft/Color analysis
From 2008.igem.org
>> Work in Progress
Analysis
Genes of the Color Pathway
The enzymes necessary to produce colored Escherichia coli colonies will be isolated from E. coli genomic DNA and Saccharomyces cerevisiae cDNA. A total of eight enzymes are needed to produce FPP, after that we will make use of BioBrick [http://partsregistry.org/Part:BBa_I742152 I742152] and [http://partsregistry.org/Part:BBa_I742161 I742161] to make sure colonies will turn red. Other colors (orange and yellow also see the Edinburgh 2007 wiki) can be produced by adding other enzymes. Of the eight enzymes we will isolate three are E. coli enzymes (atoB, idi and ispA), while the other five are S. cerevisiae enzymes (ERG8, ERG12, ERG13, MVD1 and HMG2).
The first gradient PCR have been performed on the genes, the E. coli genes all worked correctly (see lab notebook entry of August 19th), while still some work has to be done on the S. cerevisiae primers (August 20th). The next step will be to PCR the E. coli genes again, with the ideal annealing temperature, using Pfx polymerase instead of Taq. The Pfx enzyme has proofreading and so is less likely to make a mistake within the genes. The gradient PCR on S. cerevisiae cDNA will be repeated and optimized.
